Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum

Abstract

Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.

Reconstitution of Glucosylceramide Flip-Flop across Endoplasmic Reticulum

“…A key feature of this model is that GlcCer flipping for GSL synthesis occurs at the ER rather than at the Golgi complex. The low specificity of the ER phospholipid flippases suggests that these as yet unidentified transporters (14) may also be responsible for flipping GlcCer (13,15). We now report biochemical reconstitution studies to test this proposal.…”

“…TE (typically ϳ1.5 mg/ml protein determined by the micro-BCA method) was either used directly or snap-frozen and stored at Ϫ80°C. Proteoliposomes were reconstituted from a mixture of TE and Triton X-100-solubilized lipids as described (14,16,17). Briefly, chloroform stocks of egg phosphatidylcholine (3.7 mol), egg phosphatidylglycerol (0.4 mol), and NBD-lipid (0.012 mol) were combined in a glass screw-cap tube, dried under nitrogen, and solubilized in Buffer A.…”

“…Glycerol Gradient Centrifugation-TE was fractionated by velocity gradient sedimentation on a linear glycerol gradient as described previously (14,19). Briefly, 300 l of TE was loaded onto a 3.7-ml linear gradient of 10 -35% (w/v) glycerol (prepared in Buffer A).…”

Cellular organelles: how lipids get there, and back

“…Most notably, glycosphingolipids, sphingomyelin (SM), disaturated phospholipids and cholesterol are tenfold enriched in the plasma membrane as compared with the endoplasmic reticulum (ER) 1 . The ER membrane bilayer is symmetrical owing to the presence of a nonenergy-requiring, ultrarapid, translocase (A. Herrmann, Berlin, Germany) for which first purification steps were presented 2 . The ER membrane has often been compared to bacterial inner membranes.…”

“…They are excluded from retrograde transport vesicles to the ER, in contrast to unsaturated phosphatidylcholine (PtdCho) species (yellow) and are transported to the plasma membrane (arrow). Further lipid asymmetry is generated at the plasma membrane, possibly already in the Golgi, by an inward pumping aminophospholipid translocase and by outward pumping ABC transporters (2). Aminophospholipids hereby accumulate in the cytosolic bilayer leaflet (blue).…”